کلون سازی و بیان ژن هماگلوتینین ویروس آنفولانزا H9N2 در سلول Sf9
محورهای موضوعی :
ویروس شناسی
محدثه محب شاهدین
1
,
مجید مقبلی
2
,
محمد کارگر
3
,
مجتبی جعفرینیا
4
1 - گروه زیست شناسی ، واحد مرودشت ، دانشگاه آزاد اسلامی ، مرودشت ایران
2 - عضوهیئت علمی
3 - ، گروه میکروبیولوژی ، دانشکده زیست شناسی ، واحد جهرم ،دانشگاه آزاد اسلامی،
4 - گروه زیست شناسی واحد مرودشت دانشگاه آزاد اسلامی مرودشت ایران
تاریخ دریافت : 1400/08/11
تاریخ پذیرش : 1401/02/06
تاریخ انتشار : 1401/03/15
کلید واژه:
واکسن نوترکیب,
ویروس آنفولانزا,
باکولوویروس,
وکتور PfastBacDual,
چکیده مقاله :
سابقه و هدف: ویروس آنفولانزا یکی از مهمترین عوامل مرگ و میر طیور بر اثر بیماری های تنفسی است که سالیانه خسارات زیادی را به صنعت پرورش طیور در سراسردنیا وارد می‌کند.پروتین هماگلوتینین(HA) از عوامل اصلی در بیماری‌زایی این ویروس است لذا هدف از پژوهش حاضر کلون کردن و بیان این ژن در سلول Sf9 با استفاده از باکولوویروس می‌باشد.مواد و روش ها: بعد از جداسازی ژنوم ویروس آنفولانزا H9N2، ژنHA توسط پرایمرهای اختصاصی با روش هایRT-PCR و PCR تکثیر و با کمک وکتورpFastBac Dual به بکمید منتقل گردید. بکمید نوترکیب واجد ژن HA به سلول میزبانDH10Bac انتقال داده شد. با ترانسفکت کردن سلولSf9 با بکمید نوترکیب، چگونگی بیان و میزان بیان پروتین نوترکیب با روش های SDS-PAGE، western blotting و بردفورد بررسی شد.یافته ها: پروتین حاصل از بکمید نوترکیب با استفاده ازSDS PAGE بررسی و خلوص آن تائید شد. غلظت پروتین نوترکیب در محلول رویی سلول هایSf9 آلوده به ویروس نوترکیب µg/ml 138 تخمین و توسطwestern blotting تائید شد.نتیجه گیری: در تحقیق حاضر ژن HA بر روی وکتورPfastBacDual با موفقیت کلون شد و بعد از انتقال بر روی بکمید و ترانسفکت به سلولSf9 بیان مناسبی از ژن در محلول رویی سلول هایSf9 بدست آمد. با انجام آزمایش های حیوانی، این پروتین می تواند به عنوان واکسن نوترکیب بر علیه آنفولانزا H9N2 مورد استفاده قرار گیرد.
چکیده انگلیسی:
Background & Objectives: Influenza virus is one of the most important causes of death due to respiratory diseases in poultry, which causes a lot of damage to the poultry industry worldwide every year. Hemagglutinin (HA) protein is one of the main factors in the pathogenesis of this virus. The aim of this research is cloning and expression of this gene in Sf9 cell using baculovirus.Materials & Methods: After isolating of the H9N2 influenza virus genome, the HA gene was amplified by specific primers by RT-PCR and PCR methods and transferred to Bakmid using pFastBac Dual vector. The recombinant HA gene was transferred to the DH10Bac host cell. By transfecting Sf9 cells with recombinant bacmid, the expression of recombinant protein was examined by SDS-PAGE, western blotting and Bradford methods.Results: The protein obtained from recombinant Bakmid was evaluated using SDS PAGE and its purity was confirmed. The concentration of recombinant protein in the supernatant of Sf9 cells infected with recombinant virus was estimated to be 138µg / ml and confirmed by western blotting.Conclusion: In the present study, the HA gene was successfully cloned on the PfastBacDual vector. With animal experiments, this protein could be used as a recombinant vaccine candida against influenza H9N2 virus.
منابع و مأخذ:
References
Bahari P, Pourbakhsh SA, Shoushtari H, Bahmaninejad MA. Molecular characterization of H9N2 avian influenza viruses isolated from vaccinated broiler chickens in northeast Iran. Tropical animal health and production. 2015 Aug;47(6):1195-201.
Barjesteh N, O'Dowd K, Vahedi SM. Antiviral responses against chicken respiratory infections: focus on avian influenza virus and infectious bronchitis virus. Cytokine. 2020 Mar 1;127:154961..
Jawetz, M. (2016). Medical Microbiology 27 edition, Lange
Carroll, K. C., et al. (2015). Jawetz Melnick and Adelbergs Medical Microbiology 27 E, McGraw-Hill Education.
Nicholson, K. G., et al. (1998). Textbook of influenza, Blackwell Science Ltd.
Bommakanti G, Citron MP, Hepler RW, Callahan C, Heidecker GJ, Najar TA, Lu X, Joyce JG, Shiver JW, Casimiro DR, ter Meulen J. Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proceedings of the National Academy of Sciences. 2010 Aug 3;107(31):13701-6.
Adel A, Arafa A, Hussein HA, El-Sanousi AA. Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails. Research in veterinary science. 2017 Jun 1;112:132-40.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27.
Swayne, D. and D. Halvorson (2008). Influenza in Diseases of Poultry, (eds. Saif, YM, Fadly, AM, Glissom, JR, McDougald, LR, Nolan, LK & Swayne, DE) 153–184, Blackwell.
Sambrook J, Russell DW. Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harbor Protocols. 2006 Jun 1;2006(1):pdb-rot3932.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27
Schägger H. Tricine–sds-page. Nature protocols. 2006 Jun;1(1):16.
Ellebedy, A. and R. Webby (2009). "Influenza vaccines." vaccine 27: D65-D68
Kuchipudi SV, Nissly RH. Novel flu viruses in bats and cattle:“Pushing the Envelope” of Influenza Infection. Veterinary sciences. 2018 Sep;5(3):71.
Skehel, J. J. and D. C. Wiley (2000). "Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin." Annual review of biochemistry 69(1): 531-569.
Steel, J., et al. (2010). "Influenza virus vaccine based on the conserved hemagglutinin stalk domain." MBio 1(1): e00018-00010.
Vemula, S. V., et al. (2013). "Broadly protective adenovirus-based multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness." PloS one 8(4).
Hajam, I. A., et al. (2020). "Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens." Veterinary research 51(1): 1-17.
Roche X, Fredrick K, Kamata A, Okuthe S, Kone P, Wiersma L, Von Dobschuetz S, Soumare B, Makonnen Y, Morzaria S, Lubroth J. 2016–2018 Spread of H5N8 highly pathogenic avian influenza (HPAI) in sub-Saharan Africa.
Subathra M, Santhakumar P, Pardhasaradhi P, Narasu ML, Chakravarty C, Lal SK. Cloning, expression and purification of haemagglutinin and neuraminidase gene of highly pathogenic avian influenza H5N1 in Escherichia coli. Current Trends in Biotechnology and Pharmacy. 2012 Apr 1;6(2):222-8.
Bidram M, Behzadian F, Fotouhi F, Fazeli M. Cloning and prokaryotic expression of the globular head domain of hemagglutinin antigen (HA1) of influenza A (H3N2) virus in Escherichia coli and Bacillus subtilis. Archives of Medical Laboratory Sciences. 2017;3.
_||_References
Bahari P, Pourbakhsh SA, Shoushtari H, Bahmaninejad MA. Molecular characterization of H9N2 avian influenza viruses isolated from vaccinated broiler chickens in northeast Iran. Tropical animal health and production. 2015 Aug;47(6):1195-201.
Barjesteh N, O'Dowd K, Vahedi SM. Antiviral responses against chicken respiratory infections: focus on avian influenza virus and infectious bronchitis virus. Cytokine. 2020 Mar 1;127:154961..
Jawetz, M. (2016). Medical Microbiology 27 edition, Lange
Carroll, K. C., et al. (2015). Jawetz Melnick and Adelbergs Medical Microbiology 27 E, McGraw-Hill Education.
Nicholson, K. G., et al. (1998). Textbook of influenza, Blackwell Science Ltd.
Bommakanti G, Citron MP, Hepler RW, Callahan C, Heidecker GJ, Najar TA, Lu X, Joyce JG, Shiver JW, Casimiro DR, ter Meulen J. Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge. Proceedings of the National Academy of Sciences. 2010 Aug 3;107(31):13701-6.
Adel A, Arafa A, Hussein HA, El-Sanousi AA. Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails. Research in veterinary science. 2017 Jun 1;112:132-40.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27.
Swayne, D. and D. Halvorson (2008). Influenza in Diseases of Poultry, (eds. Saif, YM, Fadly, AM, Glissom, JR, McDougald, LR, Nolan, LK & Swayne, DE) 153–184, Blackwell.
Sambrook J, Russell DW. Preparation and transformation of competent E. coli using calcium chloride. Cold Spring Harbor Protocols. 2006 Jun 1;2006(1):pdb-rot3932.
Gaikwad SS, Lee HJ, Kim JY, Choi KS. Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein. Clinical and experimental vaccine research. 2019 Jan;8(1):27
Schägger H. Tricine–sds-page. Nature protocols. 2006 Jun;1(1):16.
Ellebedy, A. and R. Webby (2009). "Influenza vaccines." vaccine 27: D65-D68
Kuchipudi SV, Nissly RH. Novel flu viruses in bats and cattle:“Pushing the Envelope” of Influenza Infection. Veterinary sciences. 2018 Sep;5(3):71.
Skehel, J. J. and D. C. Wiley (2000). "Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin." Annual review of biochemistry 69(1): 531-569.
Steel, J., et al. (2010). "Influenza virus vaccine based on the conserved hemagglutinin stalk domain." MBio 1(1): e00018-00010.
Vemula, S. V., et al. (2013). "Broadly protective adenovirus-based multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness." PloS one 8(4).
Hajam, I. A., et al. (2020). "Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens." Veterinary research 51(1): 1-17.
Roche X, Fredrick K, Kamata A, Okuthe S, Kone P, Wiersma L, Von Dobschuetz S, Soumare B, Makonnen Y, Morzaria S, Lubroth J. 2016–2018 Spread of H5N8 highly pathogenic avian influenza (HPAI) in sub-Saharan Africa.
Subathra M, Santhakumar P, Pardhasaradhi P, Narasu ML, Chakravarty C, Lal SK. Cloning, expression and purification of haemagglutinin and neuraminidase gene of highly pathogenic avian influenza H5N1 in Escherichia coli. Current Trends in Biotechnology and Pharmacy. 2012 Apr 1;6(2):222-8.
Bidram M, Behzadian F, Fotouhi F, Fazeli M. Cloning and prokaryotic expression of the globular head domain of hemagglutinin antigen (HA1) of influenza A (H3N2) virus in Escherichia coli and Bacillus subtilis. Archives of Medical Laboratory Sciences. 2017;3.
